Premium
Potent lipidated antagonists to protease‐activated receptor 2 (PAR 2 )
Author(s) -
Boitano Scott,
Hoffman Justin,
Flynn Andrea,
Sherwood Cara L.,
Zhang Zhenyu,
Patek Renata,
Price Theodore J.,
Vagner Josef
Publication year - 2016
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.30.1_supplement.848.6
Subject(s) - peptidomimetic , protease , g protein coupled receptor , chemistry , signal transduction , myristoylation , biochemistry , receptor , protease activated receptor 2 , peptide , phosphorylation , enzyme , enzyme linked receptor
Protease‐activated receptor‐2 (PAR 2 ) belongs to a four‐member family of G‐Protein coupled receptors (GPCRs) that contain internal ligands exposed following exogenous or endogenous protease cleavage of the extracellular amino terminus. PAR 2 is associated with a variety of inflammatory conditions, including asthma and pain. The contributions of PAR 2 signaling to disease has been hindered by the lack of potent, efficacious antagonists, and their potential for biased‐ligand signaling. We recently demonstrated that lipid tethering of known PAR 2 peptidomimetic agonists based on the primary trypsin cleavage sequence (SLIGRL) increased their potency > 200 fold. In this study, we used lipid tethering (hexadecyl ( Hdc ) group with polyethylene glycol ( PEG ) spacers) and heterocycle (2‐aminothiazoyl; 2‐at) substitution of hexapeptide sequence derived from the primary cleavage site of kallikreins 4/16 (SSKGRS) to elucidate novel PAR 2 antagonists. Compound 562 ( C562 ), 2‐aminothiazol‐4yl‐SKGRS‐ PEG 3 ‐Hdc blocks PAR 2 Ca 2+ signaling elicited via peptidomimetics (2‐at‐LIGRL‐NH 2 ) or via asthma associated protease activation ( Alternaria alternata filtrates) in cultured human bronchial epithelial cells (16HBE14o‐). This compound was a biased‐signaling antagonist in that it had no effect on mitogen activated protein kinase (MAPK) signaling, the other major signaling pathway activated via PAR 2 . A shortened version of C562 , 2‐at‐SKGR‐ PEG 3 ‐ Hdc ( C595 ), maintained antagonistic activity against peptidomimetic activation in an in vitro physiological signaling assay (xCELLigence). C595 is closely related to the previously described potent and specific PAR 2 agonist, 2‐at‐LIGR‐ PEG 3 ‐ Hdc . Thus, we screened a series of potential PAR 2 ligands with a heterocycle serine substitute followed by four amino acids (XXGR) and the PEG 3 ‐ Hdc lipid tether. We describe several potent agonists, and one partial agonist ( C608 , 2‐at‐TIGR‐ PEG 3 ‐ Hdc ) that also acts as a potent, specific and biased signaling antagonist of PAR 2 . When used in nanomolar concentrations, C608 blocked PAR 2 ‐dependent Ca 2+ signaling via protease or peptidomimetics without effects on MAPK signaling. C562, C595 and C608 are novel pharmacological tools that can be used to evaluate the physiological consequences of PAR 2 full and biased ligand signaling. Support or Funding Information This work was primarily funded by a multi‐PI grant (NS 073664 to SB, JV and TJP) from the National Institutes of Health. Additional support for this work was from the following grants: National Institute of Health training grants (T32 HL 007249 for ANF; T32 ES 007091 for CLS), National Institutes of Health R01NS 065926 (TJP) and R01AI083403 (SB; Michael O. Daines, PI), State of Arizona Technology and Research Initiative Fund Awarded through Bio5 (JV).